1H-1,8-naphthyridin-2ones as anti proliferative compounds

ABSTRACT

The present invention relates to novel antiproliferative 1H-1, 8-naphthyridin-2-ones of the general formula (I) or pharmaceutically acceptable salts thereof: In which the variable groups are as defined herein, and their preparation and use in therapeutic treatment of disorders related to inhibition of tyrosine kinases in warm blooded animals. The compounds can overcome imatinib induced drug resistance.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national phase application under 35 U.S.C. 371claiming priority to PCT/IN2014/000777, filed Dec. 15, 2014, whichapplication claims priority to IN 2781/CHE/2014, filed Jun. 6, 2014, theteachings of which are hereby incorporated by reference in theirentireties for all purposes.

FIELD OF THE INVENTION

The present invention relates to novel antiproliferative1H-1,8-naphthyridin-2-ones of the general formula (I) orpharmaceutically acceptable salts thereof:

In which the variable groups are as defined herein, and theirpreparation and use in therapeutic treatment of disorders related toinhibition of tyrosine kinases in warm blooded animals.

BACKGROUND OF THE INVENTION

Protein tyrosine kinases are large family of proteins which plays acentral role in the regulation of several disorders, particularly in themanagement of proliferative disorders. Deregulation of tyrosine kinaseactivity has emerged as a major mechanism by which cancer cells evadenormal physiologic constraints on growth, proliferation and survival. Animportant mechanism leading to tyrosine kinase deregulation is mutation.Chronic myeloid leukaemia (CML) is a chronic myelodysplastichematopoietic stem cell disorder syndrome. 95% of the CML are resultingfrom a reciprocal translocation between chromosome-9 and chromosome-22of Philadelphia chromosome. Break point cluster region (BCR) sequencesof chromosome-22 on translocation juxtaposes with the c-ABL tyrosinekinase of chromosome-9. The fusion gene produces a 210 KDa mutantprotein in which the first exon of c-ABL has been replaced by BCRsequences, encoding either 927 or 902 amino acid. Another BCR-ABL fusionprotein of 185 KDa containing BCR sequences from exon 1 fused to exon2-11 of c-ABL, is found in 10% of adult ALL patients. The BCR-ABLchimeric gene product has a tyrosine kinase activity several fold higherthan its normal counterpart and correlates with the disease phenotype.

Tyrosine kinase forms a significant share of all onco proteins taking acentre stage as possible targets for cancer therapy. The anticancer drugGleevec/Glivec/Imatinib Mesylate (Novartis STI571) is a block busterdrug for the treatment of CML and c-kit positive metastatic GIST.Gleevec selectively and effectively inhibits the kinase activity ofBCR-ABL fusion protein, which is responsible for the constitutiveproliferative signaling. While Imatinib is therapeutically highlyeffective, with improving prospects over time for sustained remissionand potential to severely limit or eliminate disease progression andtransformation, a good number of patients either fail or respond suboptimally to Imatinib. Disease eradication may not be possible withImatinib.

Distinct patterns of resistance have evolved with the use of Imatinib,and Abl kinase mutations, which alter Imatinib binding or favour kinaseconformations inaccessible to Imatinib, Dasatinib and Nilotinib theavailable alternate Abl kinase inhibitors and restore hematologic andcytogenetic remission in the majority of patients with primary failureor acquired resistance in chronic phase. In the advanced disease andPhiladelphia chromosome (Ph)+ALL, responses are more limited and relapseis common.

ABL kinase mutations generally cluster into four main categories and areassociated with particular numbered amino acid residues: ATP bindingloop (p-loop), particularly Y253 and E255 mutants; T315 mutants; M351mutants; and activation loop (a-loop), particularly H396 mutants.Modelling of Imatinib and other kinase inhibitors with the crystalstructure of the catalytic region of the ABL kinase suggests thatmutations may interrupt critical drug contact points or induce or favoura conformation of the Abl kinase in which drug binding is reduced orprecluded. Now termed the “gatekeeper” position, mutations at threonine315 confer resistance both to Imatinib and “second generation” Ablkinase inhibitors Nilotinib and Dasatinib.

Thus there is an unmet need with regard to treatment of patients havingthe T315I mutation. Omacetaxine (homoharringtonine) is approved by FDAfor the treatment of adult patients with chronic or accelerated phasechronic myeloid leukemia (CML) with resistance and/or intolerance to twoor more tyrosine kinase inhibitors (TKIs). However, it is administeredsubcutaneously with non-specific mechanism of action. Other drugcandidates include rebastinib (WO 2008/046003) and the Ponatinib (WO2007/075869). Ponatinib (ICLUSIG) is an approved as oral drug candidateand it is developed by Ariad pharmaceuticals for the treatment ofchronic myeloid leukemia (CML) and Philadelphia chromosome-positive(Ph+) acute lymphoblastic leukemia (ALL). Ponatinib was intended totarget not only native BCR-ABL, but also its isoforms that carrymutations that confer resistance to treatment with existing tyrosinekinase inhibitors, including especially the T315I mutation for which noeffective therapy exists. However the Food and Drug Administrationtemporarily suspended sales of the drug in the U.S. in 2013 because of“the risk of life-threatening blood clots and severe narrowing of bloodvessels”. This suspension was partially lifted subsequently with revisedprescribing information, a new “Black Box Warning” and a “RiskEvaluation and Mitigation Strategy” in place.

Thus there is a need for newer selective tyrosine kinase inhibitorswhich are orally active, safer than existing therapies particularly withregard to decrease in risk of life-threatening blood clots and severenarrowing of blood vessels and efficacious against the kinase mutations,including the T315I mutant. The present invention relates to a newfamily of 1H-1,8-naphthyridin-2-ones which are potent inhibitors of Abltyrosine kinase and their mutated forms, including the T315I mutant. Thecompounds of the present invention are devoid of some of the shortcomings of the existing drug products.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows x-ray powder diffraction patterns (XRPDs) for NRC 21T

FIG. 2 shows x-ray powder diffraction patterns (XRPDs) for NRC 21Thydrochloride salt

FIG. 3 shows Anti-proliferative activity of NRC-21T on various Leukemialcell lines

FIG. 4 shows Anti-Invasive property of NRC-21T on Baf3/T315I Cell line

FIG. 5 shows Establishment of Antagonism of NRC21T in T315I inducedtumour in Nude Mice

FIG. 5A shows Establishment of Antagonism of NRC21T in T315I inducedtumour in nude mice, pictures of tumour

FIG. 5B shows Establishment of Antagonism of NRC21T in T315I inducedtumour in nude mice, pictures of spleen

FIG. 6A shows Survival time study of NRC21T

FIG. 6B shows Pictures of spleen under survival time study

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a compound of formula (I)

or a pharmaceutically acceptable salts thereof; wherein,

L1 is Linker selected from unsaturated carbon bond; preferablycarbon-carbon triple bond or carbon-carbon double bond;

L2 is a linker selected from NHC(O)—, C(O)NH—;

Ring A includes, but are not limited to substituted 5 or 6 membered arylor heteroaryl ring.

Illustrative examples of ring A groups include:

Non limiting examples of compound of formula (I) includes but are notlimited to having the following structures:

The present invention provides a process for the preparation ofcompounds of formula (I) by palladium catalyzed Sonogashira coupling of6-substituted naphthyridin-2-ones (II) with iodobenzamides (III). Theschematic representation is as follows:

Several illustrative overall synthetic approaches to the preparation oftwo key intermediates II and III, based on known transformations, areillustrated in schemes 2 to 5.

Compound of formula (I) can be prepared as per above schemes. Compoundof formula (I) can be converted into an N-oxides, or itspharmaceutically acceptable salts. For example, suitable inorganic acidsare, halogen acids, such as hydrochloric acid, sulfuric acid, orphosphoric acid. Suitable organic acids are, for example, carboxylic,phosphonic, sulfonic or sulfamic acids, for example acetic acid,propionic acid, octanoic acid, decanoic acid, dodecanoic acid, glycolicacid, lactic acid, fumaric acid, succinic acid, adipic acid, pimelicacid, suberic acid, azelaic acid, malic acid, tartaric acid, citricacid, oxalic acid, amino acids, such as arginine or lysine, maleic acid,hydroxymaleic acid, methylmaleic acid, cyclohexanecarboxylic acid,adamantanecarboxylic acid, benzoic acid, -phenoxy benzoic acid,2-acetoxy benzoic acid, salicylic acid, 4-aminosalicylic acid, phthalicacid, phenylacetic acid, mandelic acid, cinnamic acid, methane- orethane-sulfonic acid, 2-hydroxyethanesulfonic acid,ethane-1,2-disulfonic acid, benzenesulfonic acid, 2-naphthalenesulfonicacid, 1,5-naphthalene-disulfonic acid, 2-, 3-or 4-methylbenzenesulfonicacid, methylsulfuric acid, ethylsulfuric acid, dodecylsulfuric acid,N-cyclohexylsulfamic acid, N-methyl-, N-ethyl- or N-propyl-sulfamicacid, or other organic protonic acids, such as ascorbic acid.

For isolation or purification purposes it is also possible to usepharmaceutically unacceptable salts, for example picrates orperchlorates. For therapeutic use, only pharmaceutically acceptablesalts or free compounds are employed (where applicable in the form ofpharmaceutical preparations), and these are therefore preferred.

The compounds of the formula (I) or N-oxide or pharmaceuticallyacceptable salts thereof inhibit to varying degrees the receptor andnon-receptor tyrosine kinases of all, which play a role in growthregulation and transformation in mammalian cells, including human cells.The receptor tyrosine kinases may be kinases of the EGF family, e.g.ErbB2 kinase (HER-2), ErbB3 kinase, ErbB4 kinase; insulin-like growthfactor receptor kinase (IGF-1 kinase), especially members of thePDGF-receptor tyrosine kinase family, such as PDGF-α and PDGF-β receptorkinase, JAK-2, CSF-1-receptor kinase, Phosphatidylinositol 3-kinases(PI-3-kinases or PI3Ks), AKT, CDK, mTOR, Kit-receptor kinase, Flt-3,Flt-4, FGFR-1, FGFR-3, FGFR-4, c-Met, RON, c-Ret, ALK and VEGF-receptorkinase. The non-receptor tyrosine kinases may be kinases such asAbl/Bcr-abl Kinase, Arg, kinases from the Src family, c-Src kinase,c-Yes. Lck, Lyn and Fyn. The compounds of the present invention havebeen found to inhibit especially Abl/Bcr-Abl kinase, including mutantforms; Lyn and Lck kinases. The compounds of the formula (I) or N-oxideor pharmaceutically acceptable salts thereof inhibit to varying degreesthe mutant forms of Abl/Bcr-Abl kinase which include the mutants of theP-loop of the kinase i.e., L284V, G250E, Q225H, Y253F & E255K; theC-helix mutants of the kinase i.e., D276G and E279H; The ATP bindingregion mutants of the kinase i.e., V299L, T315I and F317L; SH2-contactmutant of the kinase i.e., M351T; substrate binding region mutant of thekinase i.e., F359V; the A-loop mutants of the kinase i.e., L384M, H395P,H396R and G398R; and the C-terminal lobe mutant kinase i.e., F486S.

The compounds of the present invention have been found to inhibitespecially the important mutants of abl/Bcr-Abl kinases i.e., Q252H,Y253F, M351T, H396P and more particularly the compounds of formula (I)inhibit the highly resistant form of the mutated kinase i.e., the T315Imutant.

The compounds of the present invention relates furthermore to a methodfor the treatment of a neoplastic disease or disorders dependent ontyrosine kinases especially chronic myelogenous leukemia (CML), chroniclymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), acutemyeloid leukemia (AML), myelodysplastic syndrome, melanoma, germ celltumours, gastrointestinal stromal tumour (GIST), non-small cell lungcancer (NSCLC), mastocytosis, neuroblastoma, glioblastoma, astrocytoma,hepatocellular carcinoma, renal cell cancer, breast cancer, cutaneoussystemic sclerosis, prostate and colorectal cancer and other solidtumours, diabetes remissions.

The present invention relates furthermore to a method for the treatmentof a neoplastic disease which responds to an inhibition of a proteinkinase activity, which comprises administering a compound of formula (I)or a N-oxide or a pharmaceutically acceptable salt thereof, in aquantity effective against said disease, to a warm-blooded animalrequiring such treatment.

In particular the present invention relates to a method of treatment ofproliferative disorders especially leukemia, irrespective of etiology ofthe disorder, which respond to inhibition of the aforementioned tyrosinekinases, particularly the Abl/Bcr-Abl tyrosine kinase and one or more ofits severe mutated forms. The treatment comprises administering acompound of formula (I) or an N-oxide or a pharmaceutically acceptablesalt thereof, in a quantity effective against said disease, to awarm-blooded animal requiring such treatment.

The biological efficacy of the compounds of the present invention hasbeen established by In vitro efficacy evaluation on BCR-abl positivecell line K562 and mutant cell lines Baf3/T315i, M351T, E255K and WT;Matrigel invasion assay; Determination of MTD of NRC21T; Establishmentof Antagonism of NRC21T in T315I induced tumour in Nude Mice;Establishment of Survival time of NRC21T in SCID Mice.

On the basis of these studies, the compounds of formula (I) according tothe present invention shows therapeutic efficacy especially againstdisorders dependent on TK, especially in proliferative diseases.

The present invention also relates to pharmaceutical compositionscomprising an affective amount of compound of formula (I) or a N-oxideor a pharmaceutically acceptable salt especially an amount effective inthe prevention or therapy of one of the above mentioned diseases, of theactive ingredient together with pharmaceutically acceptable carriersthat are suitable for topical, enteral, for example oral or rectal, orparental administration, and may be inorganic or organic, solid orliquid. In addition to the active ingredient(s), the pharmaceuticalcompositions of the present invention may contain one or more excipientsor adjuvants. Selection of excipients and the amounts to use may bereadily determined by the formulation scientist based upon experienceand consideration of standard procedures and reference works in thefield.

Diluents increase the bulk of a solid pharmaceutical composition, andmay make a pharmaceutical dosage form containing the composition easierfor the patient and care giver to handle. Diluents for solidcompositions include, for example, microcrystalline cellulose (e.g.Avicel®), microfine cellulose, lactose, starch, pregelatinized starch,calcium carbonate, calcium sulfate, sugar, dextrates, dextrin, dextrose,dibasic calcium phosphate dihydrate, tribasic calcium phosphate, kaolin,magnesium carbonate, magnesium oxide, maltodextrin, mannitol,polymethacrylates (e.g. Eudragit®), potassium chloride, powderedcellulose, sodium chloride, sorbitol and talc.

Solid pharmaceutical compositions that are compacted into a dosage form,such as capsules may include excipients whose functions include helpingto bind the active ingredient and other excipients together aftercompression. Binders for solid pharmaceutical compositions includeacacia, alginic acid, carbomer (e.g. carbopol), carboxymethylcellulosesodium, dextrin, ethyl cellulose, gelatin, guar gum, hydrogenatedvegetable oil, hydroxyethyl cellulose, hydroxypropyl cellulose (e.g.Klucel®), hydroxypropyl methyl cellulose (e.g. Methocel®), liquidglucose, magnesium aluminum silicate, maltodextrin, methylcellulose,polymethacrylates, povidone (e.g. Kollidon®, Plasdone®), pregelatinizedstarch, sodium alginate and starch.

The dissolution rate of a compacted solid pharmaceutical composition inthe patient's stomach may be increased by the addition of a disintegrantto the composition. Disintegrants include alginic acid,carboxymethylcellulose calcium, carboxymethylcellulose sodium (e.g.,Primellose®), colloidal silicon dioxide, croscarmellose sodium,crospovidone (e.g. Kollidon®, Polyplasdone®), guar gum, magnesiumaluminum silicate, methyl cellulose, microcrystalline cellulose,polacrilin potassium, powdered cellulose, pregelatinized starch, sodiumalginate, sodium starch glycolate (e.g. Explotab®) and starch.

Glidants can be added to improve the flowability of a non-compactedsolid composition and to improve the accuracy of dosing. Excipients thatmay function as glidants include colloidal silicon dioxide, magnesiumtrisilicate, powdered cellulose, starch, talc and tribasic calciumphosphate.

When a dosage form such as a capsule is made by the compaction of apowdered composition, the composition is subjected to pressure from apunch and dye. Some excipients and active ingredients have a tendency toadhere to the surfaces of the punch and dye, which can cause the productto have pitting and other surface irregularities. A lubricant can beadded to the composition to reduce adhesion and ease the release of theproduct from the dye. Lubricants include magnesium stearate, calciumstearate, glyceryl monostearate, glyceryl palmitostearate, hydrogenatedcastor oil, hydrogenated vegetable oil, mineral oil, polyethyleneglycol, sodium benzoate, sodium lauryl sulfate, sodium stearyl fumarate,stearic acid, talc and zinc stearate.

Flavoring agents and flavor enhancers make the dosage form morepalatable to the patient. Common flavouring agents and flavor enhancersfor pharmaceutical products that may be included in the composition ofthe present invention include maltol, vanillin, ethyl vanillin, menthol,citric acid, fumaric acid, ethyl maltol, and tartaric acid. Solidcompositions may also be dyed using any pharmaceutically acceptablecolorant to improve their appearance and/or facilitate patientidentification.

The details of the present invention are provided in the Examples givenbelow which are provided for illustrative purposes only and are notintended to limit the scope of the invention in any way.

EXPERIMENTAL SECTION Example-1 4-Methyl-N-[4-[(4-methylpiperazin-1-yl)methyl]-3-(trifluoromethyl)phenyl]-3-[2-(7-oxo-8H-1,8-naphthyridin-3-yl) ethynyl]benzamide(Development code: NRC21T)

1.1 2-Amino-5-bromo-3-iodo pyridine

To a solution of 2-amino-5-bromo pyridine in DMF, trifluoro acetic acid(1.1 equiv) was added at room temperature, followed by addition ofN-Iodo succinimide (1.1 equiv) and the reaction mixture was heated at50° C. for 180 min. After completion of the reaction, reaction mass wascooled to room temperature and the product was precipitated by addingthe reaction mixture to water. After neutralization with sodiumthiosulfate and 1N NaOH the title compound was collected by filtrationas a brown solid with 90% yield.

1.2 (E)-tert-butyl 3-(2-amino-5-bromopyridin-3-yl) acrylate

To a 2-amino-5-bromo-3-iodo pyridine (1 equiv), tert-butyl acrylate (2equiv), and isopropanol (2 equivalents), Ethyl nitrite (3 equiv) wasadded propionitrile (10 equiv) and then DMF (10 equiv). The solution wasde-oxygenated with nitrogen gas for 15 minutes. The mixture was treatedwith Pd(OAc)2 (0.1 equiv) and P(O-tol)3 (0.2 equiv) then heated to 90°C. for 16 h then filtered through a pad of silica gel. The filtrate wasconcentrated, diluted with water, extracted with ethyl acetate and theorganic layer was concentrated under vacuum below 60° C. The compoundwas collected by filtration using hexane as solvent (70% yield). ESI MSm/z 299 (100%).

1.3 6-Bromo-1H-1,8-naphthyridin-2-one

To a solution of (E)-tert-butyl 3-(2-amino-5-bromopyridin-3-yl) acrylate(1 equiv) in anhydrous methanol was treated with sodium methoxide (4.9M,5 equiv) under nitrogen gas atmosphere. The solution was heated atreflux temperature for 3 h then cooled to room temperature. The mixturewas cooled in an ice water bath and treated with water under rapidstirring to give a precipitate. The solid was filtered and washed withwater. Dried under reduced pressure to give an off-white solid (80%yield). ESI MS m/z 225 (100%).

1.4 6-(2-Trimethylsilylethynyl)-1H-1,8-naphthyridin-2-one

A mixture of 6-bromo-1H-1,8-naphthyridin-2-one (1 equiv), Pd(PPh₃)₂Cl2(0.1 equiv), CuI (0.15 equiv), (i-Pr)₂EtN (4 equiv) in propionitrile wasdeoxygenated with nitrogen gas for 15 minutes. Then trimethyl silylacetylene (2 equiv) was added and heated to 90° C. for 10 h. Afterreaction mass was filtered through a pad of silica gel at roomtemperature. The filtrate was concentrated and the compound wascollected by filtration using propionitrile solvent at 0-5° C. (60%yield).

1.5 6-Ethynyl-1H-1,8-naphthyridin-2-one

To a solution of 6-(2-trimethylsilylethynyl)-1H-1,8-naphthyridin-2-one(1 equiv) in THF (10times) was slowly added 1M TBAF solution in THF (1.1equiv) at room temperature under nitrogen gas atmosphere for 15 minutesand stirred for 15 minutes. Product formation was observed by quenchingthe reaction mass in to the water. Product was collected by filtrationat 0-5° C. For further purification, recrystallization was done inacetone solvent (80% yield).

1.6 4-Nitro-2-(trifluoromethyl) benzoic acid (SC1)

Under Nitrogen gas atmosphere, a mechanically stirred mixture of2-trifluoromethyl benzoic acid (1 equiv) and Conc.H₂SO₄ (22 equiv) wascooled in an ice bath to 0-5° C. Then fuming nitric acid (9.8 equiv) wasadded drop wise at 0-5° C. for 60 min. The ice bath was removed andstirring continued for 120 min at room temperature. After completion ofreaction the reaction mixture was poured into ice water, stirred for 60min at room temperature. Filtered the suspension, washed with chilledwater and obtained the crude title compound. To remove the regio isomerthe crude product was crystallized from water (45% yield). M.P: 137-142°C.

1.7 (4-Methylpiperazin-1-yl)-[4-nitro-2-(trifluoromethyl)phenyl]methanone (SC2)

To an ice-cooled solution of 4-nitro-2-(trifluoromethyl) benzoic acid (1equiv), CH₂Cl₂ (15times) and DMF (0.5 equiv) under nitrogen atmosphere,oxalylchloride (2 equiv) was added drop wise. After 4 hrs, the resultingsolution was concentrated in vacuum. The residue was dissolved in CH₂Cl₂and added drop wise to an ice cooled solution of N-methyl piperazine(2.1 equiv) in CH₂Cl₂. After stirring for 3 h, the mixture was dilutedwith CH₂Cl₂ and washed with water, 3 portions of 10% solution of Na₂CO₃,water and brine. The organic phase was concentrated to get the titlecompound as an oil (96% yield).

1.8 [4-Amino-2-(trifluoromethyl)phenyl]-(4-methylpiperazin-1-yl)methanone (SC3)

To a solution of (4-methylpiperazin-1-yl)-[4-nitro-2-(trifluoromethyl)phenyl]methanone (1 equiv) in methanol (3 times) was added 10% Pd/Cunder nitrogen atmosphere. Then slowly added 66% aq. ammonium formatesolution (5 equiv) at room temperature (Exothermic). After stirring for120 min, filtered through a pad of silica gel. The filtrate wasconcentrated and diluted with water, extracted with CH₂Cl₂ and washedwith water. After CH₂Cl₂ concentration compound was collected byfiltration using hexane solvent (90% yield).

1.9 4-[(4-Methylpiperazin-1-yl) methyl]-3-(trifluoromethyl) aniline(SC4)

To a solution of vitride (3 equiv) in toluene (6times) under nitrogenatmosphere was slowly added lot wise [4-amino-2-(trifluoromethyl)phenyl]-(4-methylpiperazin-1-yl)methanone (1 equiv) at room temperatureduring 2 h (exothermic). After stirring for 4 h at 65° C., slowly added8% aq. NaOH solution (12 equiv) during 1 h at room temperature.Resulting solution was stirred for 30 minutes, extracted with toluene,combined toluene layers were dried over Na₂SO₄ and concentrated.Crystallization form boiling hexane afforded the title compound (50%yield).

1.10 3-iodo-4-methyl-N-[4-[(4-methylpiperazin-1-yl)methyl]-3-(trifluoromethyl) phenyl] benzamide (SC5)

To a solution of4-[(4-methylpiperazin-1-yl)methyl]-3-(trifluoromethyl)aniline (0.7equiv) in THF (4times) under nitrogen atmosphere was added3-Iodo-4-methyl benzoyl chloride (1 equiv, prepared from the reaction of3-iodo-4-methylbenzoic acid and SOCl₂) in THF at room temperature for 30minutes followed by drop wise addition of (i-Pr)₂EtN (2 equiv) and4-DMAP (0.2 equiv). After stirring at ambient temperature for 120 min,the reaction mixture was quenched with water, extracted with ethylacetate. After drying over Na₂SO₄, concentrated the ethyl acetate layerto provide the crude product. Acetone was added to this crude productand was converted to HCl salt using IPA-HCl (85% yield).

1.11 4-Methyl-N-[4-[(4-methylpiperazin-1-yl) methyl]-3-(trifluoromethyl)phenyl]-3-[2-(7-oxo-8H-1,8-naphthyridin-3-yl) ethynyl]benzamide(Development code: NRC21T)

A mixture of3-Iodo-4-methyl-N-[4-[(4-methylpiperazin-1-yl)methyl]-3-(trifluoromethyl)phenyl]benzamide (0.8 equiv, prepared as per 1, SC5),6-ethynyl-1H-1,8-naphthyridin-2-one (1 equiv, prepared as per 1.5),Pd(PPh3)₂Cl₂ (0.1 equiv), CuI (0.15 equiv), (i-Pr)₂EtN (4 equiv), in DMFunder nitrogen atmosphere was deoxygenated with Nitrogen gas for 30minutes. Reaction mass was heated to 80° C. for 5 h. Filtered through apad of silica gel at room temperature and filtrate was concentrated,diluted with water and methanol (1:2 mixture). The compound wascollected by filtration and dried. The XRD is depicted in FIG. 1. Thecompound was subjected to salt formation with conc. HCl in methanolmedium to yield titled compound as hydrated dihydrochloride salt (70%yield). The XRD is depicted in FIG. 2.

¹HNMR (400 MHz, DMSOD₆) δ12.430 (s, 1H), 10.678 (s, 1H), 8.742 (s, 1H),8.397 (s, 1H), 8.288 (s, 1H), 8.156 (d, 2H), 7.958 (d, 2H), 7.893 (s,1H), 7.537 (d, 1H), 6.645 (d, 1H), 3.946 (bs, 2H), 3.169-3.474 (bs, 8H),2.794 (s, 3H), 2.586 (s, 3H). ESI MS m/z −560.3(M+H)⁺.

Example-2 4-Methyl-N-[4-[(4-methylpiperazin-1-yl)methyl]-3-(trifluoromethyl)phenyl]-3-[(E)-2-(7-oxo-8H-1,8-naphthyridin-3-yl) vinyl] benzamide(Development code: NRC20T)

2.1 6-[2-Trimethylsilylvinyl]-1H-1,8-naphthyridin-2-one

A mixture of 6-bromo-1H-1,8-naphthyridin-2-one (1 equiv, which wasprepared as per prepared as per-1.3), Pd (PPh₃)₂Cl₂ (0.1 equiv), CuI(0.15 equiv), (i-Pr)₂EtN (4 equiv) in DMF (15 times) was deoxygenatedwith nitrogen for 15 minutes. Then vinyl trimethyl silane (2 equiv) wasadded and heated to 125° C. for 10 h. The reaction was monitored by thinlayer chromatography. Filtered through a pad of silica gel at roomtemperature. The filtrate was concentrated and the residue was purifiedby silica gel chromatography (eluted with 20 to 30% ethylacetate/hexane) to provide the title compound as a solid (30% yield).ESI MS m/z −245.22(M+H)⁺.

2.2 4-Methyl-N-[4-[(4-methylpiperazin-1-yl) methyl]-3-(trifluoromethyl)phenyl]-3-[(E)-2-(7-oxo-8H-1,8-naphthyridin-3-yl) vinyl] benzamide(Development code: NRC20T)

A mixture of3-iodo-4-methyl-N-[4-[(4-methylpiperazin-1-yl)methyl]-3-(trifluoromethyl)phenyl]benzamide (1 equiv, prepared as per example SC5),6-[2-trimethylsilylvinyl]-1H-1,8-naphthyridin-2-one (1 equiv,), Pd(OAC)₂(0.1 equiv), P(O-tol)₃ (0.2 equiv), (i-Pr)₂EtN (4 equiv), in DMF wasdeoxygenated with Nitrogen gas for 30 minutes. Then heated to 90° C. for7 h. Filtered through a pad of silica gel at room temperature, filtratewas concentrated, and the residue was purified by silica gelchromatography (eluted with 10% methanol/methylene chloride) to providethe title compound as a solid. The compound was subjected tohydrochloride salt formation using methanol and IPA-HCl.

¹HNMR (400 MHz, DMSOD₆) δ12.283 (s, 1H), 10.772-10.802 (s, 1H), 8.822(s, 1H), 8.521 (s, 1H), 8.367 (s, 2H), 8.233 (s, 1H), 8.042 (s, 1H),7.950-7.974 (d, 1H), 7.835-7.854 (d, 1H), 7.577-7.618 (d, 1H),7.409-7.465 (d, 2H), 6.611-6.634 (d, 1H), 4.155 (bs, 2H), 3.075-3.550(bs, 8H), 2.806 (s, 3H), 2.529 (s, 3H). ESI MS m/z −562.2 (M+H)⁺.

Example-3 N-[4-Methyl-3-[2-(7-oxo-8H-1,8-naphthyridin-3-yl) ethynyl]phenyl]-4-[(4-methyl piperazin-1-yl)methyl]-3-(trifluoromethyl)benzamide (Development code: NRC19T)

3.1 Methyl 4-methyl-3-(trifluoromethyl) benzoate

To a mixture of 4-methyl-3-(trifluoromethyl) benzoic acid (1 equiv),K₂CO₃ (1.5 equiv) in acetone (15 times) was added MeI (1.5 equiv) atroom temperature. Stirring was continued for 24 h. Reaction wasmonitored by thin layer chromatography. The salts were filtered andresulting filtrate was concentrated, diluted with water, extracted withethyl acetate. Concentration of organic layer afforded the pale yellowoil (95% yield). ESI MS m/z −219 (M+H′1)⁺.

3.2 Methyl 4-(bromomethyl)-3-(trifluoromethyl) benzoate

To a solution of methyl-4-methyl-3-(trifluoromethyl) benzoate (1 equiv)in chloroform was added N-bromosuccinimide (1.1 equiv) and benzoylperoxide (0.01 equiv). The reaction mixture was heated at refluxovernight (18 h). It was then cooled to room temperature, washed withwater, dried over Na₂SO₄ and concentrated. It was purified by silica gelchromatography (eluted with 1% ethyl acetate/hexane) to provide thetitle compound (65% yield). ESI MS m/z −297.

3.3 Methyl 4-[(4-methylpiperazin-1-yl) methyl]-3-(trifluoromethyl)benzoate

To a solution of methyl 4-(bromomethyl)-3-(trifluoromethyl) benzoate (1equiv) in chloroform (6times) was added to N-methylpiperazine (3 equiv)at room temperature. The resulting solution was stirred for 3 hrs, thereaction mass was washed with water and concentrated with chloroform toget the product. (95% yield). ESI MS m/z −317 (M+H)⁺.

3.4 4-[(4-Methylpiperazin-1-yl) methyl]-3-(trifluoromethyl) benzoic acid

To a solution of methyl 4-[(4-methylpiperazin-1-yl)methyl]-3-(trifluoromethyl) benzoate (1 equiv) in ethanol (10times) wasadded 2N NaOH solution (2 equiv) at room temperature. The resultingsolution was stirred for 3 h. The reaction mixture was concentrated andacidified with aqueous 2N HCl until a white solid formed. The compoundwas collected by filtration at 0-5° C. (85% yield). ESI MS m/z −303.3(M+H)⁺.

3.5 N-(3-iodo-4-methyl-phenyl)-4-[(4-methylpiperazin-1-yl)methyl]-3-(trifluoro methyl) benzamide

To a solution of 3-iodo-4-methyl aniline (0.7 equiv) in THF (10times)under nitrogen gas atmosphere was added4-[(4-methylpiperazin-1-yl)methyl]-3-(trifluoromethyl)benzoyl chloride(1 equiv, prepared from the reaction of4-[(4-methylpiperazin-1-yl)methyl]-3-(trifluoromethyl)benzoic acid andSOCl₂) in THF (4times) at room temperature for 30 minutes followed bydrop wise addition of (i-Pr)₂EtN (4 equiv) and 4-DMAP (0.2 equiv). Afterstirring at ambient temperature for 120 min, the reaction mixture wasquenched with water and extracted with ethyl acetate. The ethyl acetatelayer was concentrated to provide the crude product. Acetone was addedto this crude product and isolated as HCl salt by employing IPA-HCl (40%yield). ESI MS m/z −518 (M+H)⁺.

3.6 N-[4-Methyl-3-[2-(7-oxo-8H-1,8-naphthyridin-3-yl) ethynyl]phenyl]-4-[(4-methyl piperazin-1-yl)methyl]-3-(trifluoromethyl)benzamide (Development code: NRC19T)

A mixture ofN-(3-iodo-4-methyl-phenyl)-4-[(4-methylpiperazin-1-yl)methyl]-3-(trifluoromethyl)benzamide (0.8 equiv, prepared as per 3.5),6-ethynyl-1H-1,8-naphthyridin-2-one (1 equiv, prepared as per 1.5),Pd(PPh3)₂Cl₂ (0.1 equiv), CuI (0.15 equiv), (i-Pr)₂EtN (4 equiv), in DMF(20times) was deoxygenated with nitrogen gas for 30 minutes. Heated to80° C. for 5 h and filtered through a pad of silica gel at roomtemperature. The filtrate was concentrated, diluted with water andstirred for 1 h. The compound was collected by filtration. Methanol wasadded to the above wet compound and the product was isolated as the HClsalt by employing IPA-HCl (40% yield).

¹HNMR (400 MHz, DMSOD₆) δ12.403 (s, 1H), 10.626 (s, 1H), 8.716-8.721 (s,1H), 8.384-8.390 (s, 1H), 8.338-8.348 (d, 2H), 8.143 (s, 1H),8.063-8.069 (s, 1H), 7.945-7.969 (d, 1H), 7.706-7.732 (d, 1H),7.342-7.364 (d, 1H), 6.636-6.659 (d, 1H), 4.114 (bs, 2H), 2.894-3.502(bs, 8H), 2.790 (s, 3H), 2.482 (s, 3H). ESI MS m/z −560.28 (M+H)⁺.

Example-4 4-Methyl-N-[3-(4-methylimidazol-1-yl)-5-(trifluoromethyl)phenyl]-3-[2-(7-oxo-8H-1,8-naphthyridin-3-yl)ethynyl]benzamideDevelopment code: NRC18T)

4.1 3-Iodo-4-methyl-N-[3-(4-methylimidazol-1-yl)-5-trifluoromethyl)phenyl] benzamide

To a solution of 3-(4-methylimidazol-1-yl)-5-(trifluoromethyl)aniline(0.7 equiv, prepared according to literature known methods) in THF undernitrogen atmosphere was added 3-Iodo-4-methyl benzoyl chloride (1 equiv,prepared from the reaction of 3-iodo-4-methylbenzoic acid and SOCl₂) inTHF at room temperature during 30 minutes followed by drop wise additionof (i-Pr)₂EtN (2 equiv) and 4-DMAP (0.2 equiv). After stirring atambient temperature over 3 h, the reaction mixture was quenched withwater. The resulting mixture was stirred at 0-5° C. for 60 min. Compoundwas collected by filtration. This compound was further purified bysilica gel chromatography (eluted with 2% methanol/chloroform) toprovide the title compound (85% yield). ESI MS m/z −486.2 (M+H)⁺.

4.2 4-Methyl-N-[3-(4-methylimidazol-1-yl)-5-(trifluoromethyl)phenyl]-3-[2-(7-oxo-8H-1,8-naphthyridin-3-yl)ethynyl]benzamide(Development code: NRC18T)

A mixture of3-iodo-4-methyl-N-[3-(4-methylimidazol-1-yl)-5-(trifluoromethyl)phenyl]benzamide (0.8 equiv, prepared as per 4.1),6-ethynyl-1H-1,8-naphthyridin-2-one (1 equiv, prepared as per 1.5),Pd(PPh3)₂Cl₂ (0.1 equiv), CuI (0.15 equiv), (i-Pr)₂EtN (4 equiv), in DMF(20 times) was deoxygenated with nitrogen gas for 30 minutes. Heated to80° C. for 5 h, filtered through a pad of silica gel at roomtemperature. The filtrate was concentrated, diluted with water, stirredfor 1 h and filtered. Acetone was added to above wet compound and theproduct was isolated as the HCl salt employing IPA-HCl (20% yield).

¹HNMR (400 MHz, DMSOD₆) δ12.440 (s, 1H), 11.034 (s, 1H), 9.668 (s, 1H),8.736-8.741 (s, 1H), 8.634 (s, 1H), 8.392-8.397 (s, 1H), 8.320 (s, 1H),8.252 (s, 1H), 7.950-8.051 (m, 4H), 7.557-7.577 (d, 1H), 6.647-6.671 (d,1H), 2.591 (s, 3H), 2.377 (s, 3H). ESI MS m/z −528.27 (M+H)⁺.

Example-5 4-Methyl-N-[3-[(4-methylpiperazin-1-yl)methyl]-5-(trifluoromethyl)phenyl]-3-[2-(7-oxo-8H-1,8-naphthyridin-3-yl)ethynyl]benzamide(Development code: NRC17T)

5.1 3-Iodo-4-methyl-N-[3-[(4-methylpiperazin-1-yl)methyl]-5-(trifluoromethyl) phenyl] benzamide

To a solution of3-[(4-methylpiperazin-1-yl)methyl]-5-(trifluoromethyl)aniline (0.7equiv, prepared according to literature methods) in THF under nitrogenatmosphere was added 3-Iodo-4-methyl benzoyl chloride (1 equiv, preparedfrom the reaction of 3-iodo-4-methylbenzoic acid and SOCl₂) in THF atroom temperature during 30 minutes followed by drop wise addition of(i-Pr)₂EtN (2 equiv) and 4-DMAP (0.2 equiv). After stirring at ambienttemperature over 3 h, the reaction mixture was quenched with water. Theresulting mixture was extracted with ethyl acetate and formed productwas isolated as HCl salt in acetone. ESI MS m/z −(M+1)⁺.

5.2 4-Methyl-N-[3-[(4-methylpiperazin-1-yl) methyl]-5-(trifluoromethyl)phenyl]-3-[2-(7-oxo-8H-1,8-naphthyridin-3-yl)ethynyl]benzamide(Development code: NRC17T)

A mixture of3-iodo-4-methyl-N-[3-[(4-methylpiperazin-1-yl)methyl]-5-(trifluoromethyl)phenyl]benzamide (0.8 equiv, prepared as per 5.1),6-ethynyl-1H-1,8-naphthyridin-2-one (1 equiv, prepared as per 1.5),Pd(PPh3)₂Cl₂ (0.1 equiv), CuI (0.15 equiv), (i-Pr)₂EtN (4 equiv), in DMF(20 times) was deoxygenated with Nitrogen gas for 30 minutes. Heated to80° C. for 5 h, filtered through a pad of silica gel at roomtemperature. The filtrate was concentrated, diluted with water, stirredfor 1 h and filtered. Methanol was added and the product was isolated asthe HCl salt employing IPA-HCl (25% yield).

¹HNMR (400 MHz, DMSOD₆) δ12.432 (s, 1H), 10.796 (s, 1H), 8.741 (s, 1H),8.399 (s, 1H), 8.322 (s, 1H), 8.222 (s, 2H), 7.964 (d, 2H), 7.814 (s,1H), 7.542-(d, 1H), 6.647 (d, 1H), 4.363 (s, 2H), 3.394 (bs, 8H), 2.818(s, 3H), 2.589 (s, 3H). ESI MS m/z −(M+1)⁺.

Example-6 N-[3,5-Bis (trifluoromethyl)phenyl]-4-methyl-3-[2-(7-oxo-8H-1,8-naphthyridin-3-yl)ethynyl]benzamidebenzamide (Development code: NRC16T)

6.1 N-[3,5-Bis (trifluoromethyl) phenyl]-3-iodo-4-methyl-benzamide

To a solution of 3,5-bis trifluoro methyl aniline (0.7 equiv) in THF(6times) under Nitrogen gas atmosphere was added 3-Iodo-4-methyl benzoylchloride (1 equiv, prepared from the reaction of 3-iodo-4-methylbenzoicacid and SOCl₂) in THF at room temperature during 30 minutes followed bydrop wise addition of (i-Pr)₂EtN (2 equiv) and 4-DMAP (0.2 equiv). Afterstirring at ambient temperature over 3 h, the reaction mixture wasquenched with water. The resulting mixture was extracted with ethylacetate and concentrated. Acetone was added to the resulting residue andformed HCl salt using IPA-HCl (85% yield). ESI MS m/z −474 (M+H)⁺.

6.2 N-[3,5-Bis (trifluoromethyl) phenyl]-4-methyl-3-[2-(7-oxo-8H-1,8-naphthyridin-3-yl) ethynyl]benzamide (Development code: NRC16T)

A mixture of N-[3,5-bis(trifluoromethyl)phenyl]-3-iodo-4-methylbenzamide(0.8 equiv, prepared as per 6.1), 6-ethynyl-1H-1,8-naphthyridin-2-one (1equiv, prepared as per 1.5), Pd(PPh3)₂Cl₂ (0.1 equiv), CuI (0.15 equiv),(i-Pr)₂EtN (4 equiv), in DMF was deoxygenated with nitrogen gas for 30minutes. Heated to 80° C. for 9 h, filtered through a pad of silica gelat room temperature. The filtrate was concentrated, diluted with water,stirred for 1 h and filtered. Methanol was added and the compound wasfiltered as HCl salt by treatment with IPA-HCl (60% yield).

¹HNMR (400 MHz, DMSOD₆) δ12.429 (s, 1H), 10.874 (s, 1H), 8.746 (s, 1H),8.545 (s, 2H), 8.402 (s, 1H), 8.223 (s, 1H), 7.956-7.979 (d, 2H), 7.839(s, 1H), 7.563-7.582 (d, 1H), 6.646-6.668 (d, 1H), 2.594 (s, 3H). ESI MSm/z −516.13 (M+H)⁺.

Example-7 N-[4-Methyl-3-[2-(7-oxo-8H-1,8-naphthyridin-3-yl) ethynyl]phenyl]-3,5-bis (trifluoro methyl)benzamide benzamide (Development code:NRC15T)

7.1 N-(3-iodo-4-methyl-phenyl)-3,5-bis (trifluoromethyl)benzamide

To a solution of 3-Iodo-4-methyl aniline (0.7 equiv) in THF (6times)under nitrogen atmosphere was added 3,5-bis trifluoromethylbenzoylchloride (1 equiv, prepared from the reaction of 3,5-bistrifluoromethylbenzoic acid and SOCl₂) in THF at room temperature for 30minutes followed by drop wise addition of (i-Pr)₂EtN (4 equiv) and4-DMAP (0.2 equiv). After stirring at ambient temperature for 3 h, thereaction mixture was quenched with water. The resulting mixture wasextracted with ethyl acetate, concentrated and the compound wascollected by adding hexane and filtration (44% yield).

7.2N-[4-Methyl-3-[2-(7-oxo-8H-1,8-naphthyridin-3-yl)ethynyl]phenyl]-3,5-Bis(trifluoro methyl)benzamide (Development code: NRC15T)

A mixture ofN-(3-iodo-4-methyl-phenyl)-3,5-bis(trifluoromethyl)benzamide (0.8 equiv,prepared as per 7.1), 6-ethynyl-1H-1,8-naphthyridin-2-one (1 equiv,prepared as per example 1.5), Pd(PPh3)₂Cl₂ (0.1 equiv), CuI (0.15equiv), (i-Pr)₂EtN (4 equiv), in DMF was deoxygenated with nitrogen gasfor 30 minutes. Heated to 65° C. for 3 h and filtered through a pad ofsilica gel at room temperature. The filtrate was diluted with water andextracted with ethyl acetate. Ethyl acetate layer was concentrated andformed HCl salt in acetone solvent.

¹HNMR (400 MHz, DMSO D₆) δ12.403 (s, 1H), 10.743 (s, 1H), 8.716 (s, 1H),8.636 (s, 2H), 8.382 (s, 2H), 8.039 (s, 1H), 7.942 (d, 1H), 7.701 (d,1H), 7.365 (d, 1H), 6.635 (d, 1H), 2.490 (s, 3H). ESI MS m/z −(M+H)⁺.

Example-8 4-Methyl-3-[2-(7-oxo-8H-1,8-naphthyridin-3-yl)ethynyl]-N-[3-(trifluoromethyl) phenyl] benzamide benzamide (Developmentcode: NRC14T)

8.1 3-Iodo-4-methyl-N-[3-(trifluoromethyl) phenyl] benzamide

To a solution of 3-trifluoromethyl aniline (0.7 equiv) in THF undernitrogen atmosphere was added 3-Iodo-4-methyl benzoyl chloride (1 equiv,prepared from the reaction of 3-iodo-4-methylbenzoic acid and SOCl₂) inTHF at room temperature during 30 minutes followed by drop wise additionof (i-Pr)₂EtN (2 equiv) and 4-DMAP (0.2 equiv). After stirring toambient temperature over 3 h, the reaction mixture was quenched in towater. The resulting mixture was extracted with ethyl acetate andconcentrated. The compound was collected by filtration using hexanesolvent (49.2% yield). ESI MS m/z −(M+1)⁺.

8.2 4-Methyl-3-[2-(7-oxo-8H-1,8-naphthyridin-3-yl)ethynyl]-N-[3-(trifluoromethyl) phenyl] benzamide (Development code:NRC14T)

A mixture of 3-iodo-4-methyl-N-[3-(trifluoromethyl)phenyl]benzamide (0.8equiv, prepared as per 8.1), 6-ethynyl-1H-1,8-naphthyridin-2-one (1equiv, prepared as per 1.5), Pd(PPh3)₂Cl₂ (0.1 equiv), CuI (0.15 equiv),(i-Pr)₂EtN (4 equiv), in DMF (20 times) was deoxygenated with nitrogengas for 30 minutes. Heated to 65° C. for 3 h, filtered through a pad ofsilica gel at room temperature and filtrate was diluted with water. Thecompound was collected by filtration and HCl salt formation.

¹HNMR (400 MHz, DMSOD₆) δ12.426 (s, 1H), 10.595 (s, 1H), 8.742 (s, 1H),8.395 (s, 1H), 8.260 (s, 1H), 8.192 (s, 1H), 8.079 (d, 1H), 7.935 (d,2H), 7.596 (t, 1H), 7.533 (d, 1H), 7.459 (d, 1H), 6.644 (d, 1H), 2.587(s, 3H). ESI MS m/z −(M+H)⁺.

Example-9N-[4-Methyl-3-[2-(7-oxo-8H-1,8-naphthyridin-3-yl)ethynyl]phenyl]-3-(trifluoromethyl)benzamide benzamide (Development code: NRC13T)

9.1 N-(3-iodo-4-methyl-phenyl)-3-(trifluoromethyl)benzamide

To a solution of 3-iodo-4-methyl aniline (0.7 equiv) in THF undernitrogen atmosphere was added 3-trifluoro methyl benzoyl chloride (1equiv, prepared from the reaction of 3-trifluoromethyl benzoic acid andSOCl₂) in THF at room temperature during 30 minutes followed by dropwise addition of (i-Pr)₂EtN (4 equiv) and 4-DMAP (0.2 equiv). Afterstirring at ambient temperature for 3 h, the reaction mixture wasquenched with water. The resulting mixture was extracted with ethylacetate, concentrated and the compound was isolated by adding n-hexane.

9.2N-[4-Methyl-3-[2-(7-oxo-8H-1,8-naphthyridin-3-yl)ethynyl]phenyl]-3-(trifluoromethyl) benzamide (Development code: NRC13T)

A mixture of N-(3-iodo-4-methyl-phenyl)-3-(trifluoromethyl)benzamide(0.8 equiv, prepared as per 9.1), 6-ethynyl-1H-1,8-naphthyridin-2-one (1equiv, prepared as per 1.5), Pd(PPh3)₂Cl₂ (0.1 equiv), CuI (0.15 equiv),(i-Pr)₂EtN (4 equiv), in DMF was deoxygenated with nitrogen gas for 30minutes. Heated to 65° C. for 3 h, filtered through a pad of silica gelat room temperature and the filtrate was diluted with water. Theresulting mixture was stirred for 1 h and the compound was collected byfiltration and HCl salt using IPA-HCl.

¹HNMR (400 MHz, DMSOD₆) δ12.400 (s, 1H), 10.523 (s, 1H), 8.718 (s, 1H),8.381 (s, 1H), 8.317 (s, 1H), 8.272 (d, 1H), 8.055 (s, 1H), 7.940 (d,2H), 7.786 (t, 1H), 7.704 (d, 1H), 7.343 (d, 1H), 6.635 (d, 1H), 2.484(s, 3H). ESI MS m/z −(M+H)⁺.

Example-10

In vitro Efficacy Evaluation:

All the experimental compounds were evaluated for in vitro efficacyemploying standard cell lines used for the screening of anti-cancercompounds.

Ponatinib was used as reference drug candidate for a comparativeefficacy and safety evaluation. A laboratory sample of the drugsubstance Ponatinib was synthesized in house employing the proceduredisclosed in U.S. Pat. No. 8,114,874.

In Vitro Studies of NRC-21T on BCR-Abl Positive Cell Line K562 andMutant Cell Lines Baf3/T315i, M351T, E255K and WT.

The experimental compounds and the standard reference drug (Ponatinib)were dissolved in cell culture medium and DMSO at a concentration of 10mM for in vitro studies. The stock solution was further diluted with thesame cell culture medium and used in concentrations of 0.1 nm to 10 μm.Cell proliferation by MTT assay was done as follows. 1000 to 10,000cells were seeded per well in 96-well plate and different concentrationsof Pon-21T ranging from 10 μm to 0.1 nM were added in triplicates. Afterincubating the cells with NRC-21T for the required time period 24-72hrs, 15 μl of 5 mg/ml MTT was added and incubated for additional 4 hoursat 37° C. and 5% CO₂. After 4 hrs, formazan crystals were dissolved insolubilizing buffer overnight at 37° C. Absorbance was measured on Elisareader at dual wavelength of 570-630-nm. By MTT assay the IC₅₀ values ofthe NRC21T were computed. IC₅₀ values obtained by MTT assay weretabulated in Table-1A & Table-1B and cells of control, Ponatinib andNRC21T was photo graphed under inverted microscope and presented. (FIG.3)

TABLE 1A IC₅₀ values obtained from MTT assay Name of the Name of theMolecule/IC50 values (nM) Cell line PONATINIB NRC21T (Example-1)Baf3/T315I 8 14 Baf3/M351T 2 2 Baf3/E255K 15 16 Baf3/WT 1 11 K562 4 7

TABLE 1B IC50 values obtained from MTT assay IC/50 values (nM) S. No.Name of the molecule K562 T315I 1. Ponatinib 4 11 2. NRC-20T (Example-2)33  155 3. NRC-19T (Example-3) 7 334 4. NRC-18T (Example-4) Not done 2285. NRC-17T (Example-5) 21  712 6. NRC-16T (Example-6) Not done >10000 7.NRC-15T (Example-7) 20  7691 8. NRC-14T (Example-8) 8 1039 9. NRC-13T(Example-9) 9 1508

Example-11

Matrigel Invasion Assay:

The in-vitro invasiveness of BaF3/T315i mutant Leukemia cancer cells inthe presence of specified concentration of NRC21T were assessed. T315icells (3×10⁵) were suspended in 300 μl of serum-free medium and placedin the upper compartment of transwell chambers pre-coated with thematrigel (Millipore, Catalog No. ECM550, USA). The lower compartment ofthe chamber was filled with 500 μl serum-medium (10% FBS) and the cellswere allowed to migrate for 72 hrs. After incubation, the cells werefixed and stained with the dye provided along with the kit andquantified using Elisa plate reader at 560 nm. In Vitro matrigel assayof T315i cells in the presence of specified concentration of NRC-21Twere represented in Table 2 and FIG. 4.

TABLE 2 Anti-Invasive property of NRC-21T on T315i cell line Percentageof Anti-invasion Cell line Test product property BaF3/T315I Ponatinib12% NRC-21T 28%

Example-12

Determination of MTD of NRC21T [the MTD Study was Carried Out as PerOECD Guidelines 420]

The study was carried out using 5 (2 Male+3 Female) Swiss Albino Miceweighing 18-30 grams. All the animals were fasted for 3 hours prior tothe oral administration of the drug. The sample was administeredimmediately to all the animals according to their body weight. Afteradministration of the experimental drug all the animals were observedfor ½ hr, 1 hr, 2 hr, 4 hours and mortality was observed for 14 days. Atthe end of 14 days, all the surviving animals were autopsied and stomachwas cut opened and observed for absorption of the drug through the GIT.

-   -   NRC21T: MTD>2000 mg·kg, p.o (Single dose 14 days observation)    -   Ponatinib: MTD=50 mg/kg, p.o (Single dose 14 days observation)

CONCLUSION

Since the MTD of NRC21T is more than 2000 mg/kg, p.o according to ICHguidelines it is inferred that NRC21T is a safer experimental drug thanPonatinib. Also NRC21T has comparable IC₅₀ values with respect toPonatinib. Thus, the experimental drug of this invention, NRC21T isestablished as superior candidate in terms of safety and efficacy.

Example-12 Establishment of Antagonism of NRC21T in T315I Induced Tumourin Nude Mice: [Clackson Etal. 2009, Cancer Cells November 6; 16(5):401-412]

The study was carried out with 18 Nude Mice (9 Male+9 Female). Weighingof Nude mice was taken initially before inoculation of cell line andmade into groups as follows:

-   -   Group-I: Positive control (3 Male+3 Female)    -   Group-II: NRC21T (3 Male+3 Female)    -   Group-III: Ponatinib (3 Male+3 Female)

The cell line was inoculated into Nude Mice subcutaneously to the righthind limb flank at a strength of 1×10⁶ cells/0.2 ml. Animals wereobserved daily for the appearance of tumour. The tumour volume wasmeasured using the formula ½ l×w² (l=length of tumour & w=width oftumour). When the mean tumour volume was recorded above 400 mm³, thetreatment with the above drugs was started. The above drugs wereadministered orally daily for 30 days. Weights of Nude Mice were takendaily before dosing and tumour measurements were done on alternativedays using digital Vernier caliper. Surviving animals were sacrificedafter the dosing was complete for 30 days and the organs (tumour withskin and spleen) were collected.

The study was depicted in FIG. 5, FIG. 5A and FIG. 5B.

Initial tumour volume No. of (Mean ± SEM) Tumour volume after treatment(Mean ± SEM)(mm³) S. No Group animals (mm³) 3^(rd) Day 5^(th) Day 7^(th)Day 9^(th) Day 11^(th) Day 13^(th) Day 15^(th) Day I Control 6 418.91 ±418.91 ± 1033.8 ± 1289.98 ± 1609.66 ± 2174.92 ± 2111.92 ± 2367.41 ± (2%Gum acacia + 51.96 51.96 234.44 291.38 427.58 678.99 360.31 384.49 2%SLS) II NRC21T 6 921.35 ± 721.18 ± 183.97 ± 106.59 ± 83.21 ± 48.88 ±10.61 ± Tumour was (200 mg/kg, p.o) 176.32 141.41 8.66 11.90 8.83 4.362.3 completely absent III Ponatinib 6 864.59 ± 574.59 ± 168.23 ± 25.59 ±22.59 ± All the mice 0 0 (50 mg/kg, p.o) 155.55 103.13 13.92 8.27 4.45were dead

Example-13

Establishment of Survival Time of NRC21T in SCID Mice [Clackson Etal.2009, Cancer Cells November 6; 16(5): 401-412]

The study was carried out using 30 SCID Mice (15 Male+15 Female). Allthe SCID Mice were injected with T315I cell Line intravenously to thetail vein at a strength of 1×10⁶ cells/0.1 ml. grouping as follows:

-   -   Group-I: Positive Control (5 Male+5 Female) (Vehicle treated)    -   Group-II: NRC21T (5 Male+5 Female) (200 mg/kg, p.o)    -   Group-III: Ponatinib (5 Male+5 Female) (10 mg/kg, p.o)

After 72 hours of injection administration of drugs to the respectivegroups were started. All the animals in the groups were administeredwith the respective drugs for 30 days. In case of mortality during thestudy spleen and liver were collected and sent for histopathology.Animals in the moribund state were sacrificed and spleen and liver werecollected and sent for histology. The study results depicted in FIG. 6Aand FIG. 6B.

Results:

Survival Time S. No. Group Mean Survival Time (Mean ± SEM) (days) 1Positive Control   20 ± 0.68 2 NRC21T 36 ± 0 3 Ponatinib 36 ± 0

We claim:
 1. A compound of formula I or an N-oxide or pharmaceuticallyacceptable salt thereof:

wherein: L1 is a linker selected from a carbon-carbon triple bond or acarbon-carbon double bond; L2 is a linker selected from —NHC(O)—,—C(O)NH—; and A is selected from the group consisting of:


2. The compound according to claim 1 or an N-oxide or pharmaceuticallyacceptable salt thereof, which compound is selected from the groupconsisting of: a.4-methyl-N-[4-[(4-methylpiperazin-1-yl)methyl]-3-(trifluoromethyl)phenyl]-3-[2-(7-oxo-8H-1,8-naphthyridin-3-yl)ethynyl]benzamide;b.4-methyl-N-[4-[(4-methylpiperazin-1-yl)methyl]-3-(trifluoromethyl)phenyl]-3-[(E)-2-(7-oxo-8H-1,8-naphthyridin-3-yl)vinyl]benzamide;c.N-[4-methyl-3-[2-(7-oxo-8H-1,8-naphthyridin-3-yl)ethynyl]phenyl]-4-[(4-methylpiperazin-1-yl)methyl]-3-(trifluoromethyl)benzamide;d.4-methyl-N-[3-(4-methylimidazol-1-yl)-5-(trifluoromethyl)phenyl]-3-[2-(7-oxo-8H-1,8-naphthyridin-3-yl)ethynyl]benzamide;e.4-methyl-N-[3-[(4-methylpiperazin-1-yl)methyl]-5-(trifluoromethyl)phenyl]-3-[2-(7-oxo-8H-1,8-naphthyridin-3-yl)ethynyl]benzamide;f.N-[3,5-bis(trifluoromethyl)phenyl]-4-methyl-3-[2-(7-oxo-8H-1,8-naphthyridin-3-yl)ethynyl]benzamide;g.N-[4-methyl-3-[2-(7-oxo-8H-1,8-naphthyridin-3-yl)ethynyl]phenyl]-3,5-bis(trifluoromethyl)benzamide;h.4-methyl-3-[2-(7-oxo-8H-1,8-naphthyridin-3-yl)ethynyl]-N-[3-(trifluoromethyl)phenyl]benzamide;i.N-[4-methyl-3-[2-(7-oxo-8H-1,8-naphthyridin-3-yl)ethynyl]phenyl]-3-(trifluoromethyl)benzamide.3. The compound according to claim 1 or a pharmaceutically acceptablesalt thereof, which compound is4-methyl-N-[4-[(4-methylpiperazin-1-yl)methyl]-3-(trifluoromethyl)phenyl]-3-[2-(7-oxo-8H-1,8-naphthyridin-3-yl)ethynyl]benzamide.4. A pharmaceutical composition comprising (i) a compound of Formula(I), or an N-oxide or pharmaceutically acceptable salt thereof; and (ii)a pharmaceutically acceptable carrier, wherein the compound of Formula(I) has the structure:

wherein: L1 is a linker selected from a carbon-carbon triple bond or acarbon-carbon double bond; L2 is a linker selected from —NHC(O)—,—C(O)NH—; and A is selected from the group consisting of:


5. A pharmaceutical composition according to claim 4, whichpharmaceutical composition comprises (i)4-methyl-N-[4-[(4-methylpiperazin-1-yl)methyl]-3-(trifluoromethyl)phenyl]-3-[2-(7-oxo-8H-1,8-naphthyridin-3-yl)ethynyl]benzamideor a pharmaceutically acceptable salt thereof; and (ii) apharmaceutically acceptable carrier.
 6. The compound according to claim1 or an N-oxide or pharmaceutically acceptable salt thereof, whichcompound is a compound of Formula (Ia):


7. The compound according to claim 1 or an N-oxide or pharmaceuticallyacceptable salt thereof, which compound is a compound of formula (Ib):